CN116445317A

CN116445317A - New streptomyces albus strain for producing puromycin and application thereof

Info

Publication number: CN116445317A
Application number: CN202211316025.3A
Authority: CN
Inventors: 段文凯; 郑玲辉; 齐欢; 张辉; 王继栋; 朱海东; 童国通; 刘雅婷
Original assignee: Hangzhou Vocational and Technical College
Current assignee: Hangzhou Vocational and Technical College
Priority date: 2022-10-26
Filing date: 2022-10-26
Publication date: 2023-07-18

Abstract

The invention provides a new strain of Streptomyces albus for producing Puromycin and application thereof, the preservation number is CGMCC25619, the classification name is Streptomyces sp, the Puromycin productivity is up to 3000mg/L, puromycin analogue Puromycin E can be produced at the same time, new precious strain resources are supplemented, and the application value is very high.

Description

New streptomyces albus strain for producing puromycin and application thereof

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a new streptomyces albus strain for producing puromycin and application thereof.

Background

Puromycin is an aminoglycoside antibiotic which can be obtained by fermentation of Streptococcus albus, is a protein synthesis inhibitor, kills gram positive bacteria, various animals and insect cells by inhibiting protein synthesis, has melting point of 175.5-177.0 ℃, optical rotation-11 (ethanol), and has chemical formula of C ₂₂ H ₂₉ N ₇ O ₅ Decomposing into 6-dimethylamine purine, O-methyl-L-tyrosine and 3-amino-3-deoxyribose in acid solution. It has a structure similar to that of the amino acid terminal gene linked to adenosine in aminoacyl tRNA molecule, so that it can be used as analogue of aminoacyl tRNA to replace some aminoacyl tRNA to enter ribosome A site and combine with extending polypeptide chain, and when the extended peptide is transferred into abnormal A site, it is easy to fall off, and in the form of peptide puromycin, it is dissociated from ribonucleoprotein early to terminate peptide chain synthesis, so that it can inhibit protein synthesis.

Puromycin is characterized by rapid cell action, typically within 2 days, killing 99% of cells that do not express the pac gene. Puromycin has an interference effect on the translation process of procaryotes and eucaryotes, has a general application in cell stable transgenic plant screening, can be used for screening specific mammal stable transfected cell strains carrying puromycin N-acetyltransferase (PAC) gene plasmids, and is mainly used as a biochemical tool for researching protein synthesis. Some have tried for tumor treatment.

The traditional preparation method of puromycin comprises a fermentation method and a chemical synthesis method, wherein the chemical synthesis method mainly uses 3-azido-adenosine as a raw material, the puromycin is prepared by 6 steps of chemical catalysis, the whole route is long, and various toxic and harmful reagents (such as TMSCl, pyridine, DCC and the like) are needed in the chemical preparation process, so that the safety coefficient in the production process is low, the environmental compatibility is low, the whole yield is not high, and the final production cost is high; the fermentation method is produced by fermentation of streptomyces albus, is more environment-friendly, environment-friendly and mild in condition, is still an effective way for preparing the puromycin, but the yield of the existing puromycin-producing streptomyces albus is very low, the puromycin content in a fermentation product is usually about 1000mg/L, and the purification process is complex and the production cost is high due to a large number of byproducts. Therefore, how to realize the production of puromycin by a more efficient and safer method is a problem to be solved at present.

Disclosure of Invention

In order to solve the problems, the invention provides a new strain of Streptomyces albus producing Puromycin and application thereof, wherein the strain is obtained by separating and purifying a mountain soil sample collected from Fengmin county of Qishan in Shaanxi by a team of inventors, and screening a large number of the mountain soil sample, the preservation number is CGMCC25619, the classification name is Streptomyces sp, the Puromycin producing capacity is up to 3000mg/L, and Puromycin analogues Puromycin E can be produced simultaneously.

In one aspect, the invention provides a novel Streptomyces albus strain with a preservation number of CGMCC 25619.

The novel Streptomyces albus strain (Streptomyces sp.HU182) has a preservation number of CGMCC25619, a preservation time of 2022, 8 months and 31 days, a preservation place of China general microbiological culture Collection center, and an address of Beijing Korea North Chen Xi Lu No.1 and 3.

Further, the nucleotide sequence of the 16S rDNA of the novel strain is shown as SEQ ID NO. 1.

In some modes, the novel strain has different culture characteristics on different culture mediums, for example, colony morphology in ISP1 culture medium is irregular and irregular in folds, the colony diameter is 0.8mm, and the strain is deep orange yellow and rich in spore production; the bacterial colony in the ISP2 culture medium is in the shape of protruding round with sawtooth-shaped white edge spores, the diameter of the bacterial colony is 1mm, the bacterial colony is dark yellow green, and the spore production is rich; the bacterial colony in the ISP3 culture medium has a shape of concentric circles, a smooth surface with a leaf edge, a diameter of 4mm, gray yellow green color and rich spore production; the colony in the ISP4 culture medium is circular, has a regular edge and a non-smooth surface, is provided with white bulges in the middle, has a diameter of 2mm, is pale yellow and has low spore production; the bacterial colony in the Chlamydia medium is circular, has irregular circular yellow pigment, has radial white outer ring, inner ring is off-white, the diameter of the bacterial colony is 0.2mm, and the bacterial colony is bright yellow and has rich spore production.

In some embodiments, the novel species can utilize carbon sources such as glucose, raffinose, xylose, sorbitol, arabinose, maltose, fructose, sucrose, galactose, inositol, rhamnose, and the like, and can utilize inorganic nitrogen sources such as glycine, aspartic acid, tyrosine, and the like.

In some embodiments, the novel bacterial species are physiologically and biochemically characterized by: gelatin liquefaction is positive (+), starch hydrolysis is positive (+), milk coagulation is positive (+), esterase tween 40 is positive (+), esculin is positive (+), nitrate reduction is positive (+), hydrogen sulfide generation is positive (+), esterase tween 60 is positive (+), cellulose utilization is negative (-), catalase is positive (+), urease is positive (+), and esterase tween 80 is positive (+).

Furthermore, the new strain can produce Puromycin analogue Puromycin E simultaneously with Puromycin production, and the structural formulas of Puromycin and Puromycin analogue Puromycin E are respectively shown as the formula (1) and the formula (2):

the novel strain provided by the invention can simultaneously produce Puromycin Puromycin and Puromycin analogue Puromycin E, and the Puromycin analogue Puromycin E can be directly converted into Puromycin Puromycin under the condition of controlling fermentation conditions.

Further, the puromycin production capacity of the novel strain reaches 3000mg/L.

In another aspect, the invention provides a process for fermentative production of puromycin by fermentation using a novel species of Streptomyces sp.HU182 as described above.

Further, the method is obtained by aerobic fermentation using a novel Streptomyces sp.HU182 strain in a nutrient medium containing assimilable carbon and nitrogen sources.

Further, the assimilable carbon source is selected from one or a combination of glucose, soluble starch, dextrin, corn starch, industrial molasses, glycerol, sucrose, sorbitol, mannitol, sorbitol, lactose, maltose syrup, crystalline maltose, preferably glucose, maltodextrin, soluble starch and combinations thereof; wherein the assimilable nitrogen source is selected from one or a combination of yeast powder, yeast extract, soybean meal, cotton seed meal, peanut meal, malt extract, peptone, beef extract, yeast extract, corn steep liquor dry powder, gluten meal, urea, ammonium salt, preferably cotton seed meal, yeast powder, malt extract or a combination thereof.

In some embodiments, the fermentation medium comprises soluble starch, maltodextrin, cotton seed meal, yeast extract, malt extract.

In some embodiments, the fermentation medium is 2% soluble starch; maltodextrin 2%; 1% of cotton seed cake powder; yeast extract 0.5%; malt extract 0.5%; mgSO4.7H2O 0.2%; naCl 0.2%; caCO3 0.2%.

In some embodiments, the fermentation is carried out at a temperature of 20 to 35 ℃, preferably 25 to 30 ℃, and a pH of 6.0 to 8.3, preferably 7.0 to 7.8, for a fermentation period of 100 to 240 hours, preferably 160 to 190 hours. Preferably, the fermentation mode is liquid submerged fermentation.

In another aspect, the invention provides the use of a novel strain of Streptomyces albus as described above for the preparation of puromycin.

Further, the puromycin yield reaches 3000mg/L.

In a further aspect, the present invention provides the use of a novel strain of Streptomyces albus as described above for the preparation of Puromycin analogue Puromycin E having the structural formula (2):

the Puromycin analogue Puromycin E can be used for producing Puromycin by a chemical synthesis method after being extracted and purified. In addition, the Puromycin analogue Puromycin E in the fermentation product of the new strain of the streptomyces albus can be converted into Puromycin through the conversion of fermentation conditions, namely the concentration of Puromycin produced by fermentation of the streptomyces albus can be higher through optimizing fermentation conditions such as fermentation medium, temperature, pH and dissolved oxygen.

The new strain provided by the invention is completely different from the known puromycin producing strain, has special physiological and biochemical characteristics, greatly improves the puromycin producing capability, has few byproducts, can simultaneously produce puromycin analogues, has low puromycin production cost, and can realize the truly high-efficient and safe puromycin production.

The novel streptomyces albus strain provided by the invention has the beneficial effects that:

1. the new strain of the streptomyces albus capable of producing puromycin at high yield is obtained by first screening;

2. the puromycin yield reaches 3000mg/L;

3. puromycin and Puromycin analogue Puromycin E can be prepared simultaneously;

4. the new precious strain resources are supplemented, and the method has high application value.

Drawings

FIG. 1 is a photograph showing colonies of the strain HU182 of example 2 on 10 media of ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, nahniko medium, nutrient agar and Bennet medium, respectively;

FIG. 2 is an HPLC chart of the fermentation broth of strain HU182 in example 5;

FIG. 3 shows the structural formula and molecular formula of PURO-1 in example 6 by MS analysis;

FIG. 4 shows hydrogen spectrum 1H-NMR of PURO-1 in example 6 by nuclear magnetic resonance spectroscopy;

FIG. 5 is an enlarged view of hydrogen spectrum 1H-NMR analyzed by nuclear magnetic resonance spectroscopy of PURO-1 in example 6;

FIG. 6 is a partial enlarged view of hydrogen spectrum 1H-NMR analyzed by nuclear magnetic resonance spectroscopy of PURO-1 in example 6;

FIG. 7 shows the carbon spectrum 13C-NMR of PURO-1 in example 6 by nuclear magnetic resonance spectroscopy;

FIG. 8 is a DEPT135 spectrum of PURO-1 of example 6 analyzed by nuclear magnetic resonance spectroscopy;

FIG. 9 is a correlation spectrum cosy of PURO-1 analyzed by nuclear magnetic resonance spectroscopy in example 6;

FIG. 10 is a heteronuclear multiple quantum relationship HMQC spectrum of PURO-1 of example 6 by nuclear magnetic resonance spectroscopy;

FIG. 11 is a graph showing the HMBC spectra of the multi-bond hydrocarbon relationship of PURO-1 in example 6, as analyzed by nuclear magnetic resonance spectroscopy;

FIG. 12 shows the structural formula and molecular formula of PURO-6 in example 6 by MS analysis;

FIG. 13 shows hydrogen 1H-NMR spectrum of PURO-6 in example 6 by nuclear magnetic resonance spectroscopy;

FIG. 14 is an enlarged view of hydrogen spectrum 1H-NMR analyzed by nuclear magnetic resonance spectroscopy of PURO-6 in example 6;

FIG. 15 is a carbon spectrum 13C-NMR spectrum of PURO-6 by nuclear magnetic resonance spectroscopy in example 6;

FIG. 16 is a DEPT135 spectrum of PURO-6 of example 6 by nuclear magnetic resonance spectroscopy;

FIG. 17 is a correlation spectrum cosy of PURO-6 in example 6 by nuclear magnetic resonance spectroscopy;

FIG. 18 is a heteronuclear multiple quantum relationship HMQC spectrum of PURO-6 of example 6 by nuclear magnetic resonance spectroscopy;

FIG. 19 is a graph showing the HMBC spectra of the multi-bond hydrocarbon relationship of PURO-6 in example 6, as analyzed by nuclear magnetic resonance spectroscopy.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto.

The invention provides a novel streptomyces albus strain capable of producing puromycin compounds, which is obtained by separating a mountain soil sample collected from Fengming town in Qishan county of Shanxi province by an inventor team, wherein the internal mark is HU182, the novel streptomyces albus strain is preserved in China general microbiological culture Collection center at 8 months 31 days of 2022, the preservation address is North Chen West road No.1 in the Chaoyang area of Beijing, the preservation number is CGMCC25619, and the classification is named streptomyces albus Streptomyces sp HU182.

EXAMPLE 1 isolation and screening of strains

Cross sampling is carried out in a mountain area delimited area of Fengming town in Qishan county of Shaanxi, 6 sampling points are randomly selected, 50g of wet soil sample is taken from each point, and natural air drying is carried out on the dry and cool places. Then, 10g of the air-dried soil sample was sufficiently ground in a mortar, 100mL (250 mL flask, 10 glass beads) of sterilized water was added thereto, and 30. Mu.L of phenol was further added thereto, and the mixture was shaken at 200rpm and 28℃for 1 hour to obtain a soil suspension. Sequentially diluting the soil suspension in 10 times of gradient, and taking 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 150. Mu.L of each of the dilutions was plated on Gao's first medium (containing 25mg/L of nalidixic acid), in an incubator at 28℃for 4-5 days, and single colonies of actinomycetes were selected. The single colony is selected on ISP2 culture medium, and purified by streaking method to obtain pure actinomycete single colony.

Obtaining 1500 strains of primary screening strains, then carrying out dibbling operation, placing the plates after dibbling at the constant temperature of 28 ℃ for 7-9 days, scraping hypha in the spore ribbon matrix of the strain by using an inoculating shovel under the aseptic condition after spores are mature, respectively inoculating the hypha into 250ml conical flasks containing 25ml of seed culture medium, and carrying out shaking culture at the temperature of 28 ℃ for 48 hours to obtain seed liquid. The seed solutions were then inoculated in 10% inoculum size into 250ml shake flasks containing 30ml fermentation medium, respectively, and shake-cultured at 28℃and 250rpm for 8 days to obtain fermentation broths.

Then adding 3 times volume of ethanol into the fermentation liquor, carrying out ultrasonic treatment for 30 minutes, sampling, detecting the puromycin content by using HPLC, using puromycin standard substance solution as a control, selecting a strain with puromycin characteristic absorption peak, and verifying the molecular weight of the characteristic absorption peak by using HPLC-MS (liquid phase mass spectrometer), wherein the molecular weight of the characteristic absorption peak is matched with that of the puromycin, thus proving that the strain can produce the puromycin. The detection proves that the strain HU182 (Streptomyces sp.HU182) can produce puromycin with high yield.

Puromycin can be detected by HPLC by the following conditions:

the chromatographic column is a C18 column, 5 μm, 4.6X1250 mm, and the column temperature is 25 ℃;

mobile phase a: water containing 5mM ammonium acetate;

mobile phase B:100% acetonitrile;

gradient procedure: 0-30 min:5% -100% (V/V) phase B; 30-33 min:100% phase B; 33-34 min:100% -5% (V/V) phase B; 34-40 min:5% phase B;

flow rate: 1mL/min;

detection wavelength: 254nm;

sample injection amount: 10-20 mu L.

EXAMPLE 2 characterization of morphology and culture of novel Streptomyces species

In this example, morphological and cultural characteristics of the highly puromycin-producing strain HU182 (Streptomyces sp. HU182) obtained in example 1 were studied, and experiments were performed with reference to the relevant contents of books such as "Manual of identification of common bacterial systems", "molecular cloning Experimental guidelines", and "Chinese pharmacopoeia". Inoculating to 10 media of ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, nahniki's medium, nutrient agar and Bennett medium respectively, culturing at 28deg.C for 7 days, observing the shape, color and other conditions of bacterial colonies, wherein the culture photographs of bacterial colonies on 10 media of ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, nahniki's medium, nutrient agar and Bennett medium are shown in FIG. 1, and include a front photograph of bacterial colony and a back photograph of bacterial colony; the culture characteristics of colonies on 10 media are shown in Table 1.

TABLE 1 culture characteristics of strain HU182 on 10 media

EXAMPLE 3 study of physiological and Biochemical characteristics of novel Streptomyces albus

This example carries out a physiological and biochemical characterization study on the highly puromycin-producing strain HU182 (Streptomyces sp. HU182) obtained in example 1.

1. Carbon source utilization test and inorganic nitrogen source utilization test:

carbon source utilization test: preparing a carbon source basic culture medium ((NH) ₄ ) ₂ SO ₄ 2.64g,KH ₂ PO ₄ 2.38g,K ₂ HPO ₄ 5.65g,MgSO ₄ ·7H ₂ O 1g，CuSO ₄ ·5H ₂ O 0.0064g，FeSO ₄ ·7H ₂ O 0.0011g，MnCl ₂ ·4H ₂ O 0.0079g,ZnSO ₄ After 0.0015g of 7H2O, 1000ml of distilled water and pH 6.5), 0.5g of each of 11 kinds of sugar was added to 100ml of mother liquor and 1.8g of agar, and placed in a 250ml Erlenmeyer flask, and 12 kinds of medium in total were set as a control medium (CK), the culture temperature was 28℃and the culture time was 7 days, and the culture results were shown in Table 2.

Inorganic nitrogen source utilization test: preparation of nitrogen basal Medium (D-glucose 1g, mgSO) ₄ ·7H ₂ O 0.05g，NaCl 0.05g，FeSO ₄ ·7H ₂ O 0.001g，K ₂ HPO ₄ 0.01g, 100ml of distilled water, pH 7.2), and 0.5g of each of three amino acids glycine, tyrosine and asparagine was added to 100ml of mother liquor and 1.8g of agar, and placed in a 250ml conical flask, and 4 kinds of medium were used as a control medium (without adding any nitrogen source), the culture temperature was 28℃and the culture time was 7 days, and the culture results were shown in Table 2.

TABLE 2 carbon and Nitrogen Source utilization of Strain HU182

Carbon source	Growth conditions	Inorganic nitrogen source	Growth conditions
				Glucose	4	Glycine (Gly)	3
Raffinose	3	Aspartic acid	2
				Xylose	4	Tyrosine	3
Sorbitol	3	CK	4
				Arabinose (Arabic sugar)	3
Maltose	4
				Fructose	4
Sucrose	3
				Galactose	4
Inositol (inositol)	3
				Rhamnose (rhamnose)	3
Ck	3

* And (3) injection: 0, no growth; 1, growth is weak; 2, the plant can grow and has a small amount of spores; 3, the growth is good, and a large number of spores are formed; 4, the best growth, rich spores.

As can be seen from table 2, strain HU182 can utilize carbon sources such as glucose, raffinose, xylose, sorbitol, arabinose, maltose, fructose, sucrose, galactose, inositol, rhamnose, etc., among which glucose, xylose, maltose, fructose, galactose are preferred carbon sources; can use inorganic nitrogen sources such as glycine, aspartic acid, tyrosine, etc.

2. Principal physiological and biochemical characterization studies

Physiological and biochemical characteristic research, including gelatin liquefaction experiments, starch hydrolysis experiments, milk coagulation experiments, esterase Tween 40 experiments, esculin experiments, nitrate reduction experiments, hydrogen sulfide generation experiments, esterase Tween 60 experiments, cellulose utilization experiments, catalase experiments, urease experiments and esterase Tween 80 experiments, is carried out by adopting a method provided by a common bacterial System identification manual. The results are shown in Table 3.

TABLE 3 physiological and biochemical characteristics of strain HU182

* And (3) injection: positive +; negative.

As can be seen from Table 3, the physiological and biochemical characteristics of the novel strain HU182 are: gelatin liquefaction is positive (+), starch hydrolysis is positive (+), milk coagulation is positive (+), esterase tween 40 is positive (+), esculin is positive (+), nitrate reduction is positive (+), hydrogen sulfide generation is positive (+), esterase tween 60 is positive (+), cellulose utilization is negative (-), catalase is positive (+), urease is positive (+), and esterase tween 80 is positive (+).

3. PH test

ISP3 was used as a basal medium, the pH of the medium was adjusted as shown in Table 4, the culture temperature was 28℃and the culture time was 7 to 10 days, and the pH conditions suitable for the growth of strain HU182 were examined, and the results are shown in Table 4.

TABLE 4 pH test of growth of strain HU182

pH	4.0	5.0	6.0	7.0	8.0	9.0	10.0	11.0	12.0
										Growth conditions	0	0	1	1	1	1	1	0	0

As can be seen from Table 4, the pH range for which strain HU182 was suitable for growth was 6.0 to 10.0, and growth was difficult at other pH.

4. Temperature test

The growth of strain HU182 at various temperatures was examined by culturing at the culture temperatures shown in Table 5 for 7 to 10 days using ISP2 medium as a basal medium, and the results are shown in Table 5.

TABLE 5 temperature test of growth of strain HU182

Temperature (. Degree. C.)	4	18	25	28	34
						Growth conditions	0	4	4	3	4

As can be seen from Table 5, strain HU182 is suitable for cultivation in the range of 18-34℃with a most preferred cultivation temperature of 28℃for the following reasons: strain HU182 grew well at 28℃with a large number of spores, and at this temperature the fermentation broth produced the highest concentration of puromycin.

5. NaCl tolerance test

ISP2 medium was used as a basal medium, naCl at the concentration shown in Table 6 was added thereto, the culture temperature was 28℃and the culture time was 8 days, and the tolerance of strain HU182 to NaCl at different concentrations was examined, and the results are shown in Table 6.

TABLE 6 tolerance of strain HU182 to NaCl

NaCl concentration%	0	1	2	3	4	5	6	7
									Growth conditions	1	1	1	1	1	0	0	0

* And (3) injection: 0, no growth; 1, growth is weak; 2, the plant can grow and has a small amount of spores; 3, the growth is good, and a large number of spores are formed; 4, growing best, and enriching spores; positive +; negative.

As can be seen from Table 6, strain HU182 is able to tolerate NaCL at concentrations below 4%.

EXAMPLE 4 16srDNA sequence analysis and Strain identification of novel Streptomyces albus species

Collecting fresh thalli of a strain to be detected, extracting a total DNA template by a Piccher method (Letters in Applied Microbiology,1989, 8:151-156) modified by a solution bacterial enzyme method, amplifying a 16S rRNA gene by using universal primers (27F and 1495R), detecting and purifying a PCR product, directly carrying out sequence determination, and sequencing by Shanghai biological engineering technology Co. The measured 16S rDNA sequence is compared with the sequences of related species and genus in GenBank database by BLAST to determine the classification status of the strain.

The 16S rDNA sequence of the strain HU182 (CGMCC 25619) is compared with related sequences in GenBank by BLAST, the sequence has very high homology with Streptomyces sp, the highest homology is more than 99 percent, meanwhile, the apparent characteristic experiment is carried out on the strain HU182 (CGMCC 25619), and the classification related parameters of the strain HU182 (CGMCC 25619) and the Streptomyces sp are very close, so the strain HU182 (CGMCC 25619) is identified as the Streptomyces sp strain.

EXAMPLE 5 preparation of puromycin by shake flask fermentation

This example uses strain HU182 provided in example 1 for fermentative preparation of puromycin,

(1) Slant culture: the yeast extract 4.0g, malt extract 10.0g, glucose 4.0g and CoCl are adopted ₂ ·6H ₂ 0.005g of O, 18.0g of agar, 1000ml of distilled water, pH value of 7.0-7.2, sterilizing at 121 ℃ for 20min, inoculating, and culturing at 28 ℃ for 6-10 days.

(2) Seed culture: seed medium composition: glucose 4.0g, malt extract 10.0g, yeast extract 4.0g, caCO ₃ 2.0g, 1000ml of distilled water, pH 7.0-7.2, packaging 30ml of distilled water in each bottle with 250ml triangular flask, washing the inclined surface Streptomyces spores with 10-15 ml of sterile water to obtain spore suspension with concentration of 1×10 ⁶ ～1×10 ⁹ And each ml. 1ml of spore suspension is added into each bottle, and the mixture is placed on a shaking table, and cultured for 48 hours at the temperature of 28 ℃ at the rotating speed of 200-250 r/min.

(3) Fermentation: fermentation medium composition: 3% of soluble starch, 3% of maltodextrin, 0.5% of yeast powder, 3% of malt extract, 0.3% of NaCl and CaCO ₃ 0.2%, pH 7.0-7.2, and sterilizing with distilled water at 121deg.C for 20min. Inoculating the seed solution into a 1L fermenter (250L) according to 5% inoculum size, culturing at 28deg.C at rotation speed of 150-250r/min, fermenting and culturing for 8 days, and detecting by HPLC, wherein HPLC diagram of fermentation broth of strain HU182 is shown in figure 2, and fermentation unit of puromycin is 2696mg/L.

EXAMPLE 6 tank fermentation to puromycin

(2) Preparing shake flask seeds: seed medium composition: glucose 4.0g, malt extract 10.0g, yeast extract 4.0g, caCO ₃ 2.0g, 1000ml of distilled water, pH 7.0-7.2, 200ml of distilled water is packed in each bottle by a 1000ml triangular flask, and then 10-15 ml of aseptic water is used for washing the inclined surface streptomycete spores to prepare spore suspension with the concentration of 1 multiplied by 10 ⁶ ～1×10 ⁹ And each ml. 2-4 ml spore suspension is added into each bottle, and the mixture is placed on a shaking table, and cultured for 48 hours at the temperature of 28 ℃ at the rotating speed of 200-250 r/min.

(3) Preparing seed tank seed liquid: the seed medium composition was the same as in example 5, and 50L of the fermentation tank was used for 30L and sterilized at 121℃for 25min. Inoculating seed liquid prepared in a seed tank into a 50L fermentation tank by adopting a differential pressure method, wherein the inoculum size is 2-3L, fermenting and culturing at 28 ℃ at the rotating speed of 150-350r/min, the ventilation rate is 0.5-1 vvm, the tank pressure is 0.05MPa and culturing for 2 days, and at the moment, the pH of the seed liquid is 6.2-7.2, and the mycelium concentration is 15-30%.

(4) Fermentation in 50L tank: the fermentation medium composition was the same as in example 5, and 15L seed was used in an amount of 8L and sterilized at 121℃for 20min. 400mL of seed liquid is inoculated into a seed tank for cultivation at 28 ℃, the rotating speed is 150-250r/min, the ventilation quantity is 0.5-1 vvm, the tank pressure is 0.05MPa, the dissolved oxygen is controlled to be not lower than 15%, the fermentation cultivation is carried out for 8 days, the tank is placed, and the fermentation unit of puromycin is 3135mg/L.

EXAMPLE 7 isolation and purification and structural characterization of fermentation products

This example was directed to the isolation, purification and structural identification of the fermentation product after 7 days of culture in the fermenter of example 6.

1. Purification and separation process of compound

Centrifuging 30L tank fermentation liquor, separating into mycelium and supernatant, soaking mycelium with 5L ethanol twice, loading HP-20 macroporous adsorbent resin on supernatant, eluting with 95% ethanol, mixing ethanol extract and concentrated eluate with silica gel, standing in a fume hood, loading into column after silica gel powder is dried, sample volume of 300mL, column loading volume of 0.75L, column specificationGradient elution (100:0, 98:2, 95:5, 90:10, 85:15, 70:30) was performed using methylene chloride/methanol as the elution phase, 1L of each gradient was collected with a conical flask having a volume of 250mL, and the collected fractions were subjected to thin layer chromatography, and were combined and concentrated to give 4 fractions I (2-8), II (9-16), III (17-25), IV (26-32).

II (9-16) passing through gel (LH-20) chromatographic columnElution with a dichloromethane/methanol (1:1, v/v) system was performed at a volume of 400mL, collected in a 10mL tube, and then according to the detection result of Thin Layer Chromatography (TLC), the following fractions were obtained: II-1 (13-23), II-2 (24-28), II-3 (29-40), performing ODS reversed phase column gradient elution (20% methanol, 30% methanol, 40% methanol, 50% methanol, 55% methanol, 70% methanol, 80% methanol, 90% methanol) with methanol as eluent, each gradient being 70mL, collecting with 10mL test tube, then obtaining the following fractions II-2-1 (37-52), II-2-2 (53-60), II-2-3 (61-final), II-2-1 (37-52), II-2-60), II-2-3 (61-final) respectively performing large preparation (15% -25% acetonitrile: 85% -75% ammonium acetate gradient), collecting with 10mL test tube, obtaining II-2-5-1 (20-26), II-2-6-1 (14-26), II-2-7-1 (18-5-26), II-2-1 (14-26), II-2-1 (18-5-26), II-2-7-1 (18-22) was combined and desalted by a further large preparation (15% -25% acetonitrile: 85% -75% pure water), collected in a 10mL test tube, and subjected to Thin Layer Chromatography (TLC) to give a compound PURO-1 (11-33,1.2 g).

I (2-8) is firstly passed through gel (LH-20) chromatographic columnElution with a dichloromethane/methanol (1:1, v/v) system was performed at a volume of 400mL, collected in a 10mL tube, and then according to the detection result of Thin Layer Chromatography (TLC), the following fractions were obtained: i-1 (7-36), ODS reversed phase column was subjected to gradient elution with methanol as eluent (50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, 100% methanol), each gradient was 200mL, collection was performed with a 10mL test tube, and then, based on the detection result of Thin Layer Chromatography (TLC), the following fractions were obtained: i-1-1 (5-15), I-1-2 (17-30). Large scale preparation of I-1-1 (5-15) (acetonitrile: pure water=12:88), collection with 10mL test tube, detection by Thin Layer Chromatography (TLC)As a result, I-1-1 (5-10) was obtained, followed by preparation (5% -15% acetonitrile: 85% -75% pure water) in large scale, collection was performed in a 10mL test tube, and according to the detection result of Thin Layer Chromatography (TLC), a compound PURO-6 (36-38, 40 mg) was obtained.

2. Structural identification

The compounds PURO-1 and PURO-6 were structurally identified by MS mass spectrometry and nuclear magnetic resonance spectrometry.

MS analysis shows that the molecular weight of the PURO-1 is 471.2230, the structural formula and the molecular formula are shown in figure 3, nuclear magnetic resonance spectroscopy analysis shows that hydrogen spectrum 1H-NMR is shown in figures 4, 5 and 6, carbon spectrum 13C-NMR is shown in figure 7, DEPT135 spectrum is shown in figure 8, correlation magnetic resonance spectrum cosy is shown in figure 9, heteronuclear multiple quantum relationship HMQC spectrum is shown in figure 10, and multi-bond hydrocarbon relationship HMBC spectrum is shown in figure 11, so that the PURO-1 is puromycin.

MS analysis shows that the molecular weight of the PURO-6 is 308.1233, the structural formula and the molecular formula are shown in figure 12, nuclear magnetic resonance spectroscopy analysis shows that hydrogen spectrum 1H-NMR is shown in figures 13 and 14, carbon spectrum 13C-NMR is shown in figure 15, DEPT135 spectrum is shown in figure 16, correlation magnetic resonance spectrum 1H-1H COSY spectrum is shown in figure 17, heteronuclear multiple quantum relationship HMQC spectrum is shown in figure 18, and multiple bond hydrocarbon relationship HMBC spectrum is shown in figure 19, so that the PURO-6 is Puromycin analogue Puromycin E.

Example 8 identification of the puromycin producing Capacity of novel Streptomyces albus

The seed medium and the fermentation medium provided in example 5 were fermented, and the conventional puromycin-producing strain and the novel strain HU182 provided in the present invention were examined for the ability to produce puromycin by shaking culture at 28℃for 8 days, and the results of the examination are shown in Table 9.

TABLE 9 comparison of puromycin-producing Capacity of different strains

As can be seen from Table 9, the novel strain HU182 provided by the invention has the puromycin-producing capability far exceeding the level of the existing strain, reaching about 3 times of the existing puromycin-producing strain, and can simultaneously produce puromycin analogues, the potency reaches more than 4 times of the existing puromycin-producing strain, the content of byproducts is less, and the cost is lower.

Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.

Sequence listing

SEQ ID NO.1

16S rDNA of Streptomyces sp.HU182:

GTGGCGGCTCTACCATGCAAGTCGACGATGAAGCCCTTCGGGGTGGATTAGTGGCGAACGGGTGA

GTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGAT

AACACCTCCACTCTCCTGGGTGGAGGTTAAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTAT

CAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACC

GGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCAC

AATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCT

TTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAG

CAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGC

GGCTTGTCACGTCGGGTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGCTAGCT

AGAGTGTGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAAC

ACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGA

ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGAACTAGGTGTTGGCGACATTCCAC

GTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAAC

TCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGA

AGAACCTTACCAAGGCTTGACATACACCGGAAAGCATCAGAGATGGTGCCCCCCTTGTGGTCGGT

GTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG

CGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGAAGACCGCCG

GGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCAC

ACGTGCTACAATGGCAGGTACAATGAGCTGCGATACCGTGAGGTGGAGCGAATCTCAAAAAGCCT

GTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATC

AGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGT

AACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCGTCGAAGGTGACGTCG

Claims

1. a novel strain of Streptomyces albus has a preservation number of CGMCC 25619.

2. The novel species of streptomyces albus of claim 1, wherein the nucleotide sequence of 16S rDNA of the novel species is shown in SEQ ID No. 1.

3. The new species of streptomyces albus of claim 2, wherein the new species produces Puromycin analogue Puromycin E simultaneously with Puromycin production, and the structural formulas of Puromycin and Puromycin analogue Puromycin E are shown in formula (1) and formula (2), respectively:

4. a new species of streptomyces albus as claimed in claim 3 wherein the puromycin producing capacity is up to 3000mg/L.

5. A process for producing puromycin by fermentation, which comprises fermenting a novel strain of Streptomyces albus as defined in any one of claims 1 to 4.

6. The method of claim 5, wherein the fermentation medium comprises a carbon source and/or a nitrogen source; the carbon source is selected from any one or more of glucose, soluble starch, dextrin, corn starch, industrial molasses, glycerol, sucrose, sorbitol, mannitol, sorbitol, lactose, maltose syrup and crystalline maltose; the nitrogen source is selected from one or more of yeast powder, yeast extract, soybean cake powder, cotton seed cake powder, peanut cake powder, malt extract, peptone, beef extract, yeast extract, corn steep liquor dry powder, gluten powder, urea and ammonium salt.

7. The method according to claim 5, wherein the fermentation is carried out at a temperature of 20 to 35℃and a pH of 6.0 to 8.3 for a period of 100 to 240 hours.

8. Use of a novel species of streptomyces albus as claimed in any one of claims 1 to 4 for the preparation of puromycin.

9. The use according to claim 8, wherein the puromycin yield is up to 3000mg/L.

10. Use of a novel strain of streptomyces albus as claimed in any one of claims 1 to 4 for the preparation of Puromycin analogue Puromycin E having the structural formula (2):